Recombinant vaccines and use thereof

ABSTRACT

The present invention relates to fusion molecules of antigens, the nucleic acids coding therefor and the use of such fusion molecules and nucleic acids. In particular, said invention relates to fusion molecules, comprising an antigen and the trans-membrane region and cytoplasmic region of a MHC molecule and/or the cytoplasmic region of a MHC or a SNARE molecule.

This application is a divisional of U.S. patent application Ser. No.13/471,606, filed on May 15, 2012, which, in turn, is a divisional ofU.S. patent application Ser. No. 10/575,640, filed on Feb. 9, 2007, nowU.S. Pat. No. 8,178,653, which is the National Stage of InternationalApplication No. PCT/EP2004/011512, filed on Oct. 13, 2004, each of whichis incorporated herein by reference in its entirety.

This application includes biological sequence information, which is setforth in an ASCII text file having the file name “VOS-120-DIV1-SEQ.txt”,created on Aug. 29, 2012, and having a file size of 80,864 bytes, whichis incorporated herein by reference.

The present invention relates to fusion molecules of antigens, to thenucleic acids coding therefor and to the use of such fusion moleculesand nucleic acids. The invention relates in particular to fusionmolecules which comprise an antigen and the transmembrane region andcytoplasmic region of an MHC molecule or the cytoplasmic region of anMHC molecule or of a SNARE molecule.

Fusion molecules of the invention can be used for a large number ofapplications, including in methods for inducing an immune response in amammal.

Antigen-specific T cell reactions are elicited by antigenic peptideswhich are bound to the binding groove of glycoproteins of the majorhistocompatibility complex (MHC), as part of the mechanism of the immunesystem in which foreign antigens are identified and a response to themis induced. The bound antigenic peptides interact with T cell receptorsand thus modulate an immune response. The antigenic peptides arenon-covalently bound to certain “binding pockets” formed by polymorphicresidues of the binding groove of the MHC protein.

MHC class II molecules are heterodimeric glycoproteins consisting of αand β chains. The α1 and β1 domains of these molecules fold together andform a peptide-binding groove. Antigenic peptides bind to the MHCmolecule through interaction between anchor amino acids on the peptideand α1 and β1 domains. The crystal structure of the human class II HLADR1 complex with an influenza virus peptide shows that the N and Cterminal ends of the bound peptide extend out of the binding groove, sothat the C terminus of the peptide lies near to the N terminus of the βchain [Brown, J. H. et al., 1993, Nature 364:33-39; Stern, L. J. et al.,1994, Nature 368:215-221]. MHC class I molecules have different domainorganizations than MHC class II molecules but generally a similarstructure with a peptide-binding site or groove which is remote from themembrane domains [cf. for example Rudensky, A. Y. et al., 1991, Nature353:622-627].

The initial step in the presentation of a foreign protein antigen isbinding of the native antigen to an antigen-presenting cell (APC). Afterbinding to APCs, antigens penetrate into the cells, either byphagocytosis, receptor-mediated endocytosis or pinocytosis. Suchinternalized antigens are located in intracellular membrane-boundvesicles called endosomes. Following endosome-lysosome fusion, theantigens are processed to small peptides by cellular proteases presentin the lysosomes. The peptides associate with the α and β chains of MHCclass II molecules within these lysosomes. These MHC class II molecules,which had previously been synthesized in the rough endoplasmicreticulum, are transported sequentially to the Golgi complexes and thento the lysosomal compartment. The peptide-MHC complex is presented onthe surface of APCs for T- and B-cell activation. Therefore, theaccessibility of proteolytic processing sites in the antigen, thestability of the resulting peptides in the lysosomes and the affinitiesof the peptides for MHC molecules are determining factors for theimmunogenicity of a specific epitope.

Recombinant vaccines have particular importance in human and veterinarymedicine as agents and medicaments for the prophylaxis and therapy ofinfectious diseases and cancers. The aim of vaccination with arecombinant vaccine is to induce a specific immune response to a definedantigen, which response has preventive or therapeutic activity againstdefined diseases.

A factor which is essential for the efficacy of a recombinant vaccine isoptimal stimulation of T lymphocytes of the immunized organism. Thus, anumber of animal-experimental investigations demonstrates that bothoptimal stimulation of CD8⁺ and CD4⁺ lymphocytes is necessary foreffective immunotherapy of tumors. The known major types of recombinantvaccines are based on recombinant proteins, synthetic peptide fragments,recombinant viruses and nucleic acid vaccines based on DNA or RNA. Inrecent years, vaccines based on DNA and RNA nucleic acids have becomeincreasingly important. However, only very poor or even no stimulationof CD4⁺ lymphocytes can be achieved with recombinant vaccines based onnucleic acids for very many aims, inter alia tumour antigens. For thisreason, a number of genetic modifications has been developed with theintention of increasing the immunogenicity of recombinant vaccines.Various methods have been tested in this connection to date, inter aliaheterogenization of immunogens by altering the primary sequence or byfusion to foreign epitopes, e.g. from bacteria or viruses [Lowenadler,B. et al., 1990, Eur. J. Immunol. 20: 1541-45; Clarke, B. E. et al.,1987, Nature 330: 381-84] and preparation of chimeric productsconsisting of the actual antigen and immunomodulatory proteins such ascytokines [Ruckert, R. et al., 1998, Eur. J. Immunol. 28: 3312-20;Harvill, E. T., J. M. Fleming, and S. L. Morrison, 1996, J. Immunol.157: 3165-70]. Although vaccines based on heterogenization induceenhanced immune responses, they have the great disadvantage thatimmunostimulation against the foreign epitope predominates and thatimmune responses against the actual vaccine target remain only moderatein some cases.

A further attractive possibility is fusion to sequences of proteinsintended to permit translocation of the protein into degrading cellcompartments. However, it is now known that these modifications lead toonly a moderate improvement in stimulation of CD4⁺ lymphocytes and toscarcely any enhancement of CD8⁺ immune responses [Wu, T. C. et al.,1995, Proc. Natl. Acad. Sci. U.S.A. 92: 11671-11675; Bonini, C. et al.,2001, J. Immunol. 166: 5250-57, Su, Z. et al., 2002, Cancer Res. 62:5041-5048].

It would thus be desirable for vaccines which distinctly increaseantigen presentation and thus immunogenicity in relation to a particularantigen to be available. It would further be desirable for it to bepossible to modify vaccines systematically in such a way that a maximumimmune response by CD4⁺ and CD8⁺ lymphocytes results, without the needto introduce foreign epitopes.

This object is achieved according to the invention by the subject matterof the claims.

It has been possible to establish according to the invention that fusionmolecules comprising antigen molecules and parts of histocompatibilityantigens show, when used as vaccines, an immunogenicity which isincreased >100-fold compared with the unmodified antigens, and thatsurprisingly both immune responses of CD4⁺ and CD8⁺ T lymphocytes areincreased in a manner not previously described.

The present invention relates in general to fusion molecules of antigenmolecules and to the use of such fusion molecules.

In one aspect, the invention relates to a fusion molecule whichcomprises an antigen and the cytoplasmic region of a chain of an MHCmolecule, or an antigen, a transmembrane region and the cytoplasmicregion of a chain of an MHC molecule. It is preferred for both thetransmembrane region and the cytoplasmic region to be derived from a MHCmolecule. In addition, the fusion molecule preferably comprises no MHCbinding domain.

The invention further relates to a fusion molecule which comprises anantigen and a chain of an MHC molecule or a part thereof, where the partcomprises at least the transmembrane region and the cytoplasmic regionof the chain of the MHC molecule. The part of the chain of an MHCmolecule preferably does not comprise the MHC binding domain or partsthereof. There is thus provided in particular a fusion molecule whichcomprises an antigen and a part of a chain of an MHC molecule, whichpart corresponds essentially to the sequence of the transmembrane regionconnected to the cytoplasmic region of an MHC molecule, where theexpression “transmembrane region connected to the cytoplasmic region”relates to the segment of a chain of an MHC molecule which starts withthe N-terminal end of the transmembrane region and terminates with theC-terminal end of the cytoplasmic region, in particular the C-terminalend of the complete chain of the MHC molecule. In this embodiment, theconnection of the transmembrane region to the cytoplasmic regioncorresponds to the naturally occurring connection between these regions.

The invention further provides a fusion molecule which comprises anantigen and a chain of an MHC molecule or a part thereof, where the partessentially lacks the complete N-terminal extracellular domains of theMHC molecule.

In a particularly preferred embodiment, the fusion molecules of theinvention consist of a fusion of an antigen, where appropriate with aleader sequence at its N-terminal end, to a transmembrane region,preferably a transmembrane region of a chain of an MHC molecule, at theC-terminal end of the antigen and of a cytoplasmic region of a chain ofan MHC molecule at the C-terminal end of the transmembrane region.

In a particularly preferred embodiment, the fusion molecules of theinvention comprise a leader sequence, preferably a peptide sequencehaving the properties of a secretion signal which is able in particularto control translocation of a protein or peptide through a membrane. Itis possible to use as leader sequence the secretion signal of any type Itransmembrane protein, where the expression “type I transmembraneprotein” relates to those transmembrane proteins whose C terminus islocated in the cytoplasm. In a particular embodiment, the leadersequence is derived from a chain of an MHC molecule. The leader sequenceis preferably located at the N-terminal end of the fusion molecules ofthe invention.

In a further aspect, the invention relates to a fusion molecule whereessentially the complete N-terminal extracellular domains of an MHCmolecule are replaced by an antigen having a leader sequence at itsN-terminal end.

It is preferred in a fusion molecule of the invention for the antigen tobe covalently connected at its N terminus to the C terminus of a leadersequence, and the C terminus of the antigen molecule is connected to theN terminus of the transmembrane region which in turn is connected at theC terminus to the N terminus of the cytoplasmic region of an MHCmolecule.

Thus, the fusion molecule of the invention preferably has the followingarrangement: N terminus leader sequence/antigen/transmembraneregion/cytoplasmic region C terminus.

In a particularly preferred embodiment, the fusion molecule of theinvention consists essentially of the leader sequence, the antigen, thetransmembrane region and the cytoplasmic region.

In a particularly preferred embodiment, the antigen is a peptide,polypeptide or protein, and the fusion molecule of the invention is aprotein or polypeptide.

In one embodiment, a plurality of antigens which may be identical ordifferent are present in the fusion molecule of the invention, i.e. atleast 2, preferably 2 to 10, more preferably 2 to 5, even morepreferably 2 to 3, in particular 2, antigens. These multiply coupledantigens may be present separate from one another or in series one afterthe other, where appropriate separated by a linker, as tandemconstructs. It is preferred for an immune response to various antigensto be induced thereby on administration.

The antigen may be complete or truncated, i.e. it contains only a partof the natural protein or polypeptide which serves as antigen.

The leader sequence and/or the transmembrane region of the fusionmolecules of the invention are preferably derived from MHC molecules, inparticular of class I or II. It is more preferred for the leadersequence and/or the transmembrane region and/or the cytoplasmic regionof the fusion molecules of the invention to be derived from MHCmolecules, in particular of class I or II.

It is also possible according to the invention for one or more,preferably flexible, linker sequences (connecting sequences) to bepresent in the fusion molecule, possibly being located between theleader sequence and the antigen, between the antigen and thetransmembrane region and/or between the transmembrane region and thecytoplasmic region. It is preferred according to the invention for alinker sequence to comprise about 7 to 20 amino acids, more preferablyabout 8 to 16 amino acids, and in particular about 8 to 12 amino acids.

The linker sequence in fusion molecules of the invention is preferablyflexible and thus does not hold the peptide connected therewith in asingle, unwanted conformation. The linker preferably comprises inparticular amino acids having small side chains, such as glycine,alanine and serine, in order to make flexibility possible. The linkersequence preferably comprises no proline residue, which might inhibitthe flexibility.

In a further embodiment, the leader sequence, the antigen, thetransmembrane region and/or the cytoplasmic region are connectedtogether directly without a linker.

The leader sequence preferably has the sequence shown in SEQ ID NO: 2 ora sequence derived therefrom, or is encoded by the sequence shown in SEQID NO: 1 or a sequence derived therefrom. The transmembrane-cytoplasmicregion preferably has the sequence shown in SEQ ID NO: 4 or 6 or asequence derived therefrom, or is encoded by the sequence shown in SEQID NO: 3 or 5 or a sequence derived therefrom.

In further preferred embodiments, the transmembrane-cytoplasmic or theexclusively cytoplasmic region is derived from sequence-related MHCmolecules (inter alia HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-DRa,HLA-DRb, HLA-DQa, HLA-DQb, HLA-DPa, HLA-DPb, CD1a, CD1b, CD1c).Preferred transmembrane-cytoplasmic regions have a sequence selectedfrom the group consisting of the sequences depicted in SEQ ID NO: 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and sequencesderived therefrom. In further embodiments, the exclusively cytoplasmicregions have a sequence selected from the group consisting of thesequences depicted in SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, and sequences derived therefrom. Further embodimentsalso provide for the use of varied sequences, e.g. modified ororthologous sequences from different organisms. Sequences particularlypreferred in this connection are those having at the C-terminal end ahomology of more than 60% with the sequences shown in SEQ ID NO: 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42.

In a particularly preferred embodiment, the fusion molecule of theinvention comprises the amino acid sequence shown in SEQ ID NO: 12 or14, or a sequence derived therefrom.

The invention further relates to a fusion molecule comprising an antigenand a SNARE protein (in particular Cis-golgi SNARE p28, VTI1b, membrin,pallidin, syntaxin-5, syntaxin-6, syntaxin-7, syntaxin-8, syntaxin-10,syntaxin-10a, syntaxin-11, syntaxin-12, syntaxin-17, VAMP-2, VAMP-3,VAMP-4, VAMP-7, VAMP8, VTI1-a-beta, XP350893, LIPS (SEQ ID NO: 43-63))or a sequence which comprises one or more SNARE motifs. Targetedtransport of the antigen into a defined compartment (e.g. lysosomes andendosomes) is possible by fusing an antigen to a SNARE protein or aSNARE motif (preferably at the C terminus of the SNARE protein ormotif). A further possibility with such a targeted transport is forimmunogenic epitopes of the antigen to be generated and presented in acompartment, as can be established experimentally.

SNARE proteins are membrane-associated proteins whose common feature isthe SNARE motif which comprises 60-70 amino acids. SNARE proteins arefunctionally involved in the transport and fusion of vesicles in thecell. Eukaryotic organisms have a large number of different SNAREproteins which are associated with different vesicle membranes in thecell (inter alia endosomal, lysosomal, Golgi, plasma membranes). Thecytoplasmic regions of the SNARE proteins have a dual function. Firstly,they serve as trafficking signals (address labels) which specify thedestination of the protein and of the associated membrane. Secondly, thedomains may contribute through hetero- and homoassociation (joiningtogether) to fusion of different vesicles (e.g. endosomes withlysosomes).

It is also possible according to the invention for the SNARE-antigenfusion molecules to comprise linker sequences between the SNARE portionand the antigen portion. Also included in relation to the antigen andthe linker sequence of the SNARE-antigen fusion molecules are all theembodiments described above. A linker in relation to the SNARE-antigenfusion molecules preferably comprises 80-120 amino acids. In aparticular embodiment, the linker comprises a transmembrane region. Theinvention thus relates to fusion molecules which comprise a SNAREprotein or a SNARE motif fused to an antigen or a transmembrane regionand an antigen. Such fusion molecules are shown for example in FIG. 7.

In a further aspect, the invention relates to nucleic acids andderivatives thereof which code for the fusion molecules described aboveand are preferably able to express these fusion molecules. The term“nucleic acid” hereinafter also includes derivatives thereof.

In a particularly preferred embodiment, the nucleic acid which codes fora fusion molecule of the invention comprises the nucleic acid sequenceshown in SEQ ID NO: 11 or 13, or a sequence derived therefrom.

The invention also relates to host cells which comprise a nucleic acidof the invention.

The host cell may moreover comprise a nucleic acid which codes for anHLA molecule. In one embodiment, the host cell expresses the HLAmolecule endogenously. In a further embodiment, the host cell expressesthe HLA molecule recombinantly. The host cell is preferablynon-proliferative. In a preferred embodiment, the host cell is anantigen-presenting cell, in particular a dendritic cell, a monocyte or amacrophage.

In a further aspect, the invention relates to a pharmaceuticalcomposition, in particular a vaccine, which comprises one or more of thefusion molecules of the invention and/or one or more of the nucleicacids coding therefor and/or one or more of the host cells of theinvention.

In a further aspect, the invention provides a method for increasing theamount of MHC/peptide complexes in a cell, where the method comprisesthe provision of a fusion molecule of the invention or of a nucleic acidcoding therefor for the cell. The cell is preferably present in a livingcreature, and the method comprises administering a fusion molecule ofthe invention or a nucleic acid coding therefor to the living creature.In a preferred embodiment, the cell is an antigen-presenting cell, inparticular a dendritic cell, a monocyte or a macrophage.

In a further aspect, the invention provides a method for increasing thepresentation of cell surface molecules on cells which are able topresent antigens (such as B cells and macrophages, generally called“APC”). The antigen-presenting activity of such cells is enhanced byproviding a fusion molecule of the invention or a nucleic acid codingtherefor for the cells. Such an enhancement of the antigen-presentingactivity in turn preferably enhances the primary activation of T cells,in particular of CD4⁺ and CD8⁺ lymphocytes, which respond to theantigen. The cell is preferably present in a living creature, and themethod comprises administering a fusion molecule of the invention or anucleic acid coding therefor to the living creature.

In a further aspect, the invention provides a method for inducing animmune response in a living creature, where the method comprises theadministration of a fusion molecule of the invention and/or a nucleicacid coding therefor and/or a host cell of the invention to the livingcreature.

In a further aspect, the invention provides a method for stimulating oractivating T cells, especially CD4⁺ and CD8⁺ lymphocytes, in vitro or ina living creature, in particular a patient, where the method comprisesthe provision for the T cells or administration to the living creatureof a fusion molecule of the invention and/or a nucleic acid codingtherefor and/or a host cell of the invention. Such a stimulation oractivation is preferably expressed in an expansion, cytotoxic reactivityand/or cytokine release by the T cells.

A further aspect provides a method for the treatment, vaccination orimmunization of a living creature, where the method comprises theadministration a fusion molecule of the invention and/or a nucleic acidcoding therefor and/or a host cell of the invention to the livingcreature. In this connection, the antigens employed in the fusionmolecule of the invention or the nucleic acid coding therefor are inparticular those which are known to be effective without the alterationaccording to the invention for the intended treatment, vaccination orimmunization.

The methods described above are particularly suitable for a treatment orprophylaxis of infectious diseases caused for example by bacteria orviruses. In particular embodiments, the antigen used according to theinvention is derived from an infectious agent such as hepatitis A, B, C,HIV, mycobacteria, malaria pathogens, SARS pathogens, herpesvirus,influenzavirus, poliovirus or from bacterial pathogens such as chlamydiaand mycobacteria. A particularly beneficial application of the presentinvention is in cancer immunotherapy or vaccination, where there is inparticular enhancement of activation of tumor antigen-reactive T cells,thus improving the prospects for T-cell immunotherapy or vaccinationagainst tumor cells.

In specific embodiments, the antigen used according to the invention isselected from the group consisting of the following antigens: p53,preferably encoded by the sequence shown in SEQ ID NO: 66, ART-4, BAGE,ss-catenin/m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA,CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V,Gap100, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE,LDLR/FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 orMAGE-A12, MAGE-B, MAGE-C, MART-1/melan-A, MC1R, myosin/m, MUC1, MUM-1,-2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor bcr-abL Pml/RARa,PRAME, proteinase-3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3,SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL/AML1, TPI/m, TRP-1, TRP-2,TRP-2/INT2, TPTE and WT, preferably WT-1, in particular encoded by thesequence shown in SEQ ID NO: 65.

DETAILED DESCRIPTION OF THE INVENTION

The terms “domain” or “region” relate to a particular part of an aminoacid sequence which can preferably be connected to a specific functionor structure. For example, the α and β polypeptides of an MHC class IImolecule have two domains, α1, α2, and β1, β2, respectively, atransmembrane region and a cytoplasmic region. In a similar manner, theα chain of MHC class I molecules has three domains, α1, α2 and α3, atransmembrane region and a cytoplasmic region.

In one embodiment, the complete domain or region is included in aselection of the sequence of a particular domain or region for deletionor incorporation into a fusion molecule of the invention. In order toensure this, the sequence of the relevant domain or region can beextended in order to comprise parts of a linker or even parts of theadjacent domain or region. The term “essentially” in relation to adomain or region is to be understood in this sense.

The term “transmembrane region” relates to the part of a protein whichessentially accounts for the portion present in a cellular membrane andpreferably serves to anchor the protein in the membrane. A transmembraneregion is preferably according to the invention an amino acid sequencewhich spans the membrane once. However, it is also possible in certainembodiments to use a transmembrane region which spans the membrane morethan once. The transmembrane region will generally have 15-25 preferablyhydrophobic uncharged amino acids which assume for example an α-helicalconformation. The transmembrane region is preferably derived from aprotein selected from the group consisting of MHC molecules,immunoglobulins, CD4, CD8, the CD3 ζ chain, the CD3 γ chain, the CD3 δchain and the CD3 ε chain.

The transmembrane region typically consists in the case of the α and βchains of the MHC class II molecule of about 20 hydrophobic amino acidswhich are connected to the carboxy-terminal end of the antigen. Theseresidues allow the protein to span the membrane. The transmembraneregion terminates with about 6-32 residues which comprise thecytoplasmic tail at the carboxy-terminal end of each of these chains. Ithas been shown that these transmembrane and cytoplasmic regions can bereplaced by sequences which signal a GPI binding, and that the chimericGPI-anchored class II molecules are membrane-bound (Wettstein, D. A., J.J. Boniface, P. A. Reay, H. Schild and M. M. Davis, 1991, J. Exp. Med.174: 219-228). Such embodiments are encompassed by the term“transmembrane region” according to the invention. GPI-bound membraneanchor domains have been defined in a number of proteins, includingdecay-accelerating factor (DAF), CD59 and human placental alkalinephosphatase (HPAP) (Wettstein, D. A., J. J. et al., 1991, J. Exp. Med.174:219-228). For example, the 38 carboxy-terminal amino acids of HPAPare sufficient for functioning as signal sequence for GPI binding. Ifthe DNA sequence coding for this domain is connected to a secretedmolecule, such as the soluble part of the MHC class II α or β chain,there is formation of a membrane-bound chimeric molecule (Wettstein, D.A. et al., 1991, J. Exp. Med. 174: 219-228), and a method of this typecan be employed to anchor fusion molecules of the invention to a cellmembrane.

The term “major histocompatibility complex” and the abbreviation “MHC”relate to a complex of genes which occurs in all vertebrates. Thefunction of MHC proteins or molecules in signaling between lymphocytesand antigen-presenting cells in normal immune responses involves thembinding peptides and presenting them for possible recognition by T-cellreceptors (TCR). MHC molecules bind peptides in an intracellularprocessing compartment and present these peptides on the surface ofantigen-presenting cells to T cells. The human MHC region, also referredto as HLA, is located on chromosome 6 and comprises the class I regionand the class II region.

The term “MHC class I” or “class I” relates to the majorhistocompatibility complex class I proteins or genes. Within the humanMHC class I region there are the HLA-A, HLA-B, HLA-C, HLA-E, HLA-F,CD1a, CD1b and CD1c subregions.

The class I α chains are glycoproteins having a molecular weight ofabout 44 kDa. The polypeptide chain has a length of somewhat more than350 amino acid residues. It can be divided into three functionalregions: an external, a transmembrane and a cytoplasmic region. Theexternal region has a length of 283 amino acid residues and is dividedinto three domains, α1, α2 and α3. The domains and regions are usuallyencoded by separate exons of the class I gene. The transmembrane regionspans the lipid bilayer of the plasma membrane. It consists of 23usually hydrophobic amino acid residues which are arranged in an αhelix. The cytoplasmic region, i.e. the part which faces the cytoplasmand which is connected to the transmembrane region, typically has alength of 32 amino acid residues and is able to interact with theelements of the cytoskeleton. The α chain interacts withβ2-microglobulin and thus forms α-β2 dimers on the cell surface.

The term “MHC class II” or “class II” relates to the majorhistocompatibility complex class II proteins or genes. Within the humanMHC class II region there are the DP, DQ and DR subregions for class IIα chain genes and β chain genes (i.e. DPα, DPβ, DQα, DQβ, DRα and DRβ).

Class II molecules are heterodimers each consisting of an α chain and aβchain. Both chains are glycoproteins having a molecular weight of 31-34kDa (a) or 26-29 kDA (β). The total length of the α chains varies from229 to 233 amino acid residues, and that of the β chains from 225 to 238residues. Both α and β chains consist of an external region, aconnecting peptide, a transmembrane region and a cytoplasmic tail. Theexternal region consists of two domains, α1 and α2 or β1 and β2. Theconnecting peptide is respectively 13 and 9 residues long in α and βchains. It connects the two domains to the transmembrane region whichconsists of 23 amino acid residues both in α chains and in β chains. Thelength of the cytoplasmic region, i.e. the part which faces thecytoplasm and which is connected to the transmembrane region, variesfrom 3 to 16 residues in α chains and from 8 to 20 residues in β chains.

The term “chain of an MHC molecule” relates according to the inventionto the α chain of an MHC class I molecule or to the α and β chains of anMHC class II molecule. The α chains of an MHC class I molecule, fromwhich the fusion molecules of the invention can be derived, comprise theHLA-A, -B and -C α chains. The α chains of an MHC class II molecule,from which the fusion molecules of the invention may be derived,comprise HLA-DR, -DP and -DQ α chains, in particular HLA-DR1, HLA-DR2,HLA-DR4, HLA-DQ1, HLA-DQ2 and HLA-DQ8 α chains and, in particular, αchains encoded by DRA*0101, DRA*0102, DQA1*0301 or DQA1*0501 alleles.The β chains of an MHC class II molecule, from which the fusionmolecules of the invention may be derived, comprise HLA-DR, -DP and -DQβ chains, in particular HLA-DR1, HLA-DR2, HLA-DR4, HLA-DQ1, HLA-DQ2 andHLA-DQ8 β chains and, in particular, β chains encoded by DRB1*01,DRB1*15, DRB1*16, DRB5*01, DQB1*03 and DQB1*02 alleles.

The term “MHC binding domain” relates to the “MHC class I bindingdomain” and “MHC class II binding domain”.

The term “MHC class I binding domain” relates to the region of an MHCclass I molecule or of an MHC class I chain which is necessary forbinding to an antigenic peptide. An MHC class I binding domain is formedmainly by the α1 and α2 domains of the MHC class I α chain. Although theα3 domain of the α chain and β2-microglobulin do not represent essentialparts of the binding domain, they are presumably important forstabilizing the overall structure of the MHC class I molecule andtherefore the term “MHC class I binding domain” preferably includesthese regions. An MHC class I binding domain can also be essentiallydefined as the extracellular domain of an MHC class I molecule,distinguishing it from the transmembrane and cytoplasmic regions.

The term “MHC class II binding domain” relates to the region of an MHCclass II molecule or of an MHC class II chain which is necessary forbinding to an antigenic peptide. An MHC class II binding domain ismainly formed by the α1 and β1 domains of the MHC class II α and βchains. The α2 and β2 domains of these proteins are, however, presumablyalso important for stabilizing the overall structure of the MHC bindinggroove, and therefore the term “MHC class II binding domain” accordingto the invention preferably includes these regions. An MHC class IIbinding domain can also be defined essentially as the extracellulardomain of an MHC class II molecule, distinguishing it from thetransmembrane and cytoplasmic domains.

The exact number of amino acids in the various MHC molecule domains orregions varies depending on the mammalian species and between geneclasses within a species. When selecting the amino acid sequence of aparticular domain or region, maintenance of the function of the domainor region is much more important than the exact structural definition,which is based on the number of amino acids. The skilled worker is alsoaware that the function can also be maintained if rather less than thecomplete amino acid sequence of the selected domain or region is used.

The term “antigen” relates to an agent against which an immune responseis to be generated. The term “antigen” includes in particular proteins,peptides, polysaccharides, nucleic acids, especially RNA and DNA, andnucleotides. The term “antigen” also includes derivatized antigens assecondary substance which becomes antigenic—and sensitizing—only throughtransformation (e.g. intermediately in the molecule, by completion withbody protein), and conjugated antigens which, through artificialincorporation of atomic groups (e.g. isocyanates, diazonium salts),display a new constitutive specificity. In a preferred embodiment, theantigen is a tumor antigen, i.e. a constituent of cancer cells which maybe derived from the cytoplasm, the cell surface and the cell nucleus, inparticular those antigens which are produced, preferably in largequantity, intracellularly or as surface antigens on tumor cells.Examples are carcinoembryonic antigen, α1-fetoprotein, isoferritin andfetal sulfoglycoprotein, α2-H-ferroprotein and γ-fetoprotein and variousviral tumor antigens. In a further embodiment, the antigen is a viralantigen such as viral ribonucleoproteins or envelope proteins. Inparticular, the antigen or peptides thereof should be presented by MHCmolecules and thus be able to modulate, in particular, activate, cellsof the immune system, preferably CD4⁺ and CD8⁺ lymphocytes, inparticular by modulating the activity of a T-cell receptor, and thuspreferably induce T cell proliferation.

The term “MHC/peptide complex” relates to a non-covalent complex of thebinding domain of an MHC class I or MHC class II molecule and of an MHCclass I or MHC class II binding peptide.

The term “MHC binding peptide” or “binding peptide” relates to a peptidewhich binds to an MHC class I and/or an MHC class II molecule. In thecase of class I MHC/peptide complexes, the binding peptides typicallyhave a length of 8-10 amino acids, although longer or shorter peptidesmay be active. In the case of class II MHC/peptide complexes, thebinding peptides typically have a length of 10-25 amino acids and inparticular of 13-18 amino acids, although longer and shorter peptidesmay be active.

Fusion molecules of the invention and the nucleic acids coding thereforcan generally be prepared by recombinant DNA techniques such aspreparation of plasmid DNA, cleavage of DNA with restriction enzymes,ligation of DNA, transformation or transfection of a host, cultivationof the host and isolation and purification of the expressed fusionmolecule. Such methods are known and described for example in Sambrooket al., Molecular Cloning (2nd edition, 1989).

DNA coding for the antigen can be obtained by isolating DNA from naturalsources or by known synthetic methods such as the phosphate triestermethod; cf., for example, Oligonucleotide Synthesis, IRL Press (M. J.Gait, editor, 1984). Synthetic oligonucleotides can also be preparedwith the aid of commercially available automatic oligonucleotidesynthesizers.

The proportions of MHC molecules in the fusion molecules of theinvention suitably correspond, in relation to the amino acid sequence,to naturally occurring MHC molecules from humans, mice or other rodentsor other mammals or are derivatives thereof.

DNA sources coding for MHC proteins are known, such as humanlymphoblastoid cells. After isolation, the gene coding for the MHCmolecule, or an interesting part thereof, can be amplified by polymerasechain reaction (PCR) or other known methods. Suitable PCR primers foramplifying the gene for the MHC peptide can attach restriction sites tothe PCR product.

It is preferred according to the invention to prepare DNA constructswhich comprise nucleic acid sequences coding for the leader sequence,the transmembrane region and the cytoplasmic region, and which comprisea restriction cleavage site between the leader sequence and thetransmembrane region, so that essentially any nucleotide sequence codingfor an interesting antigen can be incorporated into the construct.

In a preferred method for preparing fusion molecules of the invention,DNA sequences are disposed in such a way that the C-terminal end of theleader sequence is linked to the N-terminal end of the antigen, theC-terminal end of the antigen is linked to the N-terminal end of thetransmembrane region, and the C-terminal end of the transmembrane regionis linked to the N-terminal end of the cytoplasmic region. As discussedabove, restriction cleavage sites are preferably incorporated betweenthe end of the leader sequence and the start of the transmembraneregion, so that essentially any nucleic acid which codes for aninteresting antigen can be linked to the nucleic acid sequence for thetransmembrane region.

An expressed fusion molecule of the invention may be isolated andpurified in a manner known per se. Typically, the culture medium will becentrifuged and the supernatant will then be purified by affinity orimmunoaffinity methods comprising the use of monoclonal antibodies whichbind to the expressed fusion molecule. The fusion molecule may alsocomprise a sequence which assists purification, e.g. a 6×His tag.

The ability of a fusion molecule of the invention to modulate theactivity of a T-cell receptor (including inactivation of T-cellresponses) can easily be determined by an in vitro assay. Typically, Tcells are provided for the assays by transformed T-cell lines, such asT-cell hybridomas or T cells which are isolated from a mammal such as ahuman or a rodent such as a mouse. Suitable T-cell hybridomas are freelyavailable or can be prepared in a manner known per se. T cells can beisolated in a manner known per se from a mammal; cf., for example,Shimonkevitz, R. et al., 1983, J. Exp. Med. 158: 303.

A suitable assay for determining whether a fusion molecule of theinvention is able to modulate the activity of T cells takes place asfollows by steps 1-4 hereinafter. T cells suitably express a markerwhich can be assayed and indicates the T-cell activation or modulationof T-cell activity after activation. Thus, the mouse T-cell hybridomaDO11.10, which expresses interleukin-2 (IL-2) on activation, can beused. IL-2 concentrations can be measured in order to determine whethera specific presenting peptide is able to modulate the activity of thisT-cell hybridoma. A suitable assay of this type is carried out by thefollowing steps:

1. T cells are obtained for example from an interesting T-cell hybridomaor by isolation from a mammal.2. The T cells are cultivated under conditions which permitproliferation.3. The growing T cells are brought into contact with antigen-presentingcells which in turn have been brought into contact with a fusionmolecule of the invention or with a nucleic acid coding therefor.4. The T cells are assayed for a marker, e.g. IL-2 production ismeasured.

The T cells used in the assays are incubated under conditions suitablefor proliferation. For example, a DO11.10 T-cell hybridoma is suitablyincubated in complete medium (RPMI 1640, supplemented with 10% FBS,penicillin/streptomycin, L-glutamine and 5×10⁻⁵ M 2-mercaptoethanol) atabout 37° C. with 5% CO₂. Serial dilutions of the fusion molecule of theinvention can be assayed. T-cell activation signals are provided byantigen-presenting cells which have been loaded with the suitableantigenic peptide.

As an alternative to measuring an expressed protein such as IL-2, it ispossible to determine the modulation of T-cell activation suitably bychanges in the proliferation of antigen-dependent T cells, as measuredby known radiolabeling methods. For example, a labeled (such astritiated) nucleotide can be introduced into an assay culture medium.The introduction of such a labeled nucleotide into the DNA serves asmeasurand for T-cell proliferation. This assay is unsuitable for T cellsnot requiring antigen presentation for growth, such as T-cellhybridomas. The assay is suitable for measuring the modulation of T-cellactivation by fusion molecules in the case of untransformed T cellsisolated from mammals.

The ability of a fusion molecule of the invention to induce an immuneresponse, including making it possible to vaccinate against a targetdisease, can be determined simply by an in vivo assay. For example, afusion molecule of the invention or a nucleic acid coding therefor canbe administered to a mammal such as a mouse, and blood samples be takenfrom the mammal at the time of the first administration and severaltimes at periodic intervals thereafter (such as 1, 2, 5 and 8 weeksafter administration of the fusion molecule or of the nucleic acidcoding therefor). Serum is obtained from the blood samples and assayedfor the appearance of antibodies resulting from the immunization.Antibody concentrations can be determined. In addition, T lymphocytescan be isolated from the blood or from lymphatic organs and befunctionally assayed for reactivity to the antigen or epitopes derivedfrom the antigen. All the readout systems known to the skilled worker,inter alia proliferation assay, cytokine secretion, cytotoxic activity,tetramer analysis, can be used in this connection.

Methods of the invention for inducing an immune response, includingvaccination of a living creature against a target disease, can be usedin combination with known methods for inducing an immune response. Forexample, a fusion molecule of the invention or a nucleic acid codingtherefor can be administered to a living creature in an arrangement orcombination with administration of a vaccine composition in order toenhance or prolong the desired effect of such a vaccine composition.

The term “derived” means according to the invention that a particularentity, in particular a particular sequence, is present in the objectfrom which it is derived, in particular an organism or molecule. In thecase of nucleic acid and amino acid sequences, especially particularsequence regions, “derived” additionally means that the relevant nucleicacid or amino acid sequence is derived, consistent with the definitionshereinafter, from a nucleic acid or amino acid sequence which is presentin the object. Thus, the expression “sequence or region derived from anMHC molecule” means that the sequence or region is present in an MHCmolecule or is derived, consistent with the definitions hereinafter,from a sequence or region which is present in an MHC molecule.

A nucleic acid is according to the invention preferably deoxyribonucleicacid (DNA) or ribonucleic acid (RNA). Nucleic acids include according tothe invention genomic DNA, cDNA, mRNA, recombinantly prepared andchemically synthesized molecules. A nucleic acid may according to theinvention be in the form of a molecule which is single stranded ordouble stranded and linear or closed covalently to form a circle.

A sequence derived from a nucleic acid sequence or the expression“sequence derived from a nucleic acid sequence” relates according to theinvention to homologous sequences and derivatives of the formersequence.

Homologous nucleic acid sequences display according to the invention atleast 40%, in particular at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% and preferably at least 95%, at least 98 or atleast 99% identity of the nucleotides.

A nucleic acid is “homologous” to another nucleic acid in particularwhen the two sequences of the complementary strands are able tohybridize with one another and enter into a stable duplex, thehybridization preferably taking place under conditions which permitspecific hybridization between polynucleotides (stringent conditions).Stringent conditions are described for example in Molecular Cloning: ALaboratory Manual, J. Sambrook et al., editors, 2nd edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 or CurrentProtocols in Molecular Biology, F. M. Ausubel et al., editors, JohnWiley & Sons, Inc., New York, and relate for example to hybridization at65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02%polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH₂PO₄ (pH 7),0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15 M sodiumcitrate, pH 7. After the hybridization, the membrane onto which the DNAhas been transferred is for example washed in 2×SSC at room temperatureand then in 0.1-0.5×SSC/0.1×SDS at temperatures of up to 68° C.

“Derivative” of a nucleic acid means according to the invention thatsingle or multiple nucleotide substitutions, deletions and/or additionsare present in the nucleic acid. The term “derivative” also includes inaddition chemical derivatization of a nucleic acid on a base, a sugar orphosphate of a nucleotide. The term “derivative” also includes nucleicacids which comprise non-naturally occurring nucleotides and nucleotideanalogs.

The nucleic acids described by the invention are preferably isolated.The term “isolated nucleic acid” means according to the invention thatthe nucleic acid (i) has been amplified in vitro, for example bypolymerase chain reaction (PCR), (ii) has been produced recombinantly bycloning, (iii) has been purified, for example by cleavage andfractionation by gel electrophoresis, or (iv) has been synthesized, forexample by chemical synthesis. An isolated nucleic acid is a nucleicacid which is available for manipulation by recombinant DNA techniques.

Nucleic acids which code for fusion molecules can according to theinvention be alone or in combination with other nucleic acids,especially heterologous nucleic acids. In preferred embodiments, anucleic acid is functionally connected to expression control sequencesor regulatory sequences which may be homologous or heterologous inrelation to the nucleic acid. A coding sequence and a regulatorysequence are “functionally” connected together if they are linkedtogether covalently in such a way that expression or transcription ofthe coding sequence is under the control or under the influence of theregulatory sequence. If the coding sequence is to be translated into afunctional protein and where there is a functional connection of aregulatory sequence to the coding sequence, induction of the regulatorysequence leads to transcription of the coding sequence without theoccurrence of a shift in reading frame in the coding sequence or of aninability of the coding sequence to be translated into the desiredprotein or peptide.

The term “expression control sequence” or “regulatory sequence” includesaccording to the invention promoters, enhancers and other controlelements which control the expression of a gene. In particularembodiments of the invention, the expression control sequences can beregulated. The exact structure of regulatory sequences may varyspecies-dependently or cell type-dependently, but generally includes5′-non-transcribed and 5′-non-translated sequences which are involved ininitiating transcription and translation, respectively, such as TATAbox, capping sequence, CAAT sequence and the like. In particular,5′-non-transcribed regulatory sequences include a promoter region whichincludes a promoter sequence for transcriptional control of thefunctionally connected gene. Regulatory sequences may also includeenhancer sequences or activator sequences located upstream.

In a preferred embodiment, the nucleic acid is according to theinvention a vector, where appropriate having a promoter which controlsthe expression of a nucleic acid, e.g. of a nucleic acid which codes fora fusion molecule of the invention. In a preferred embodiment, thepromoter is a T7, T3 or SP6 promoter.

The term “vector” is used in this connection in its most general meaningand includes any of the intermediate vehicles for a nucleic acid whichmake it possible, for example, for the nucleic acid to be introducedinto prokaryotic and/or into eukaryotic cells and, where appropriate, beintegrated into a genome. Such vectors are preferably replicated and/orexpressed in the cell. An intermediate vehicle may be adapted forexample for use in electroporation, in microprojectile bombardment, inliposomal administration, in transfer with the aid of agrobacteria or ininsertion via DNA or RNA viruses. Vectors include plasmids, phagemids,bacteriophages or viral genomes.

The nucleic acids which code for a fusion molecule of the invention canbe employed for transfection of host cells. Nucleic acids mean in thisconnection both recombinant DNA and RNA. Recombinant RNA can be preparedby in vitro transcription from a DNA template. It can moreover bemodified before application by stabilizing sequences, capping andpolyadenylation.

The term “host cell” relates according to the invention to any cellwhich can be transformed or transfected with an exogenous nucleic acid.The term “host cells” includes according to the invention prokaryotic(e.g. E. coli) or eukaryotic (e.g. dendritic cells, B cells, CHO cells,COS cells, K562 cells, yeast cells and insect cells). Mammalian cellsare particularly preferred, such as cells from humans, mice, hamsters,pigs, goats and primates. The cells may be derived from a large numberof tissue types and include primary cells and cell lines. Specificexamples include keratinocytes, peripheral blood leukocytes, bone marrowstem cells and embryonic stem cells. In further embodiments, the hostcell is an antigen-presenting cell, in particular a dendritic cell, amonocyte or macrophage. A nucleic acid may be present in the host cellin a single or in multiple copies and is, in one embodiment, expressedin the host cell.

The term “expression” is used according to the invention in its mostgeneral meaning and includes the production of RNA or of RNA andprotein. It also includes partial expression of nucleic acids. Inaddition, the expression may be transient or stable. Preferredexpression systems in mammalian cells include pcDNA3.1 and pRc/CMV(Invitrogen, Carlsbad, Calif.), which comprise a selectable marker suchas a gene which confers resistance to G418 (and thus makes selection ofstably transfected cell lines possible), and the enhancer-promotersequences of cytomegalovirus (CMV).

A nucleic acid coding for a fusion molecule of the invention may alsoinclude a nucleic acid sequence which codes for an MHC molecule,preferably for an HLA molecule. The nucleic acid sequence which codesfor an MHC molecule may be present on the same expression vector as thenucleic acid which codes for the fusion molecule, or the two nucleicacids may be present on different expression vectors. In the lattercase, the two expression vectors can be cotransfected into a cell.

A sequence derived from an amino acid sequence or the expression“sequence derived from an amino acid sequence” relates according to theinvention to homologous sequences and derivatives of the formersequence.

Homologous amino acid sequences exhibit according to the invention atleast 40%, in particular at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% and preferably at least 95%, at least 98 or atleast 99% identity of the amino acid residues.

“Derivatives” of a protein or polypeptide or of an amino acid sequencein the sense of this invention include amino acid insertion variants,amino acid deletion variants and/or amino acid substitution variants.

Amino acid insertion variants include amino- and/or carboxy-terminalfusions, and insertions of single or multiple amino acids in aparticular amino acid sequence. In amino acid sequence variants with aninsertion, one or more amino acid residues are introduced into apredetermined site in an amino acid sequence, although random insertionwith suitable screening of the resulting product is also possible. Aminoacid deletion variants are characterized by deletion of one or moreamino acids from the sequence. Amino acid substitution variants aredistinguished by at least one residue in the sequence being deleted andanother residue being inserted in its stead. The modifications arepreferably present at positions in the amino acid sequence which are notconserved between homologous proteins or polypeptides. Amino acids arepreferably replaced by others having similar properties, such ashydrophobicity, hydrophilicity, electronegativity, volume of the sidechain and the like (conservative substitution). Conservativesubstitutions relate for example to replacement of one amino acid byanother, with both amino acids being listed in the same grouphereinafter:

1. small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr(Pro, Gly)2. negatively charged residues and their amides: Asn, Asp, Glu, Gln3. positively charged residues: His, Arg, Lys4. large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys)5. large aromatic residues: Phe, Tyr, Trp.

Three residues are put in parentheses because of their particular rolein protein architecture. Gly is the only residue without a side chainand thus confers flexibility on the chain. Pro has an unusual geometrywhich greatly restricts the chain. Cys can form a disulfide bridge.

The amino acid variants described above can easily be prepared with theaid of known peptide synthesis techniques such as, for example, by solidphase synthesis (Merrifield, 1964) and similar methods or by recombinantDNA manipulation. Techniques for introducing substitution mutations atpredetermined sites in DNA which has a known or partially known sequenceare well known and include, for example, M13 mutagenesis. Manipulationof DNA sequences to prepare proteins having substitutions, insertions ordeletions and the general recombinant methods for expression of proteinsfor example in a biological system (such as mammalian, insect, plant andviral systems) are described in detail for example in Sambrook et al.(1989).

“Derivatives” of proteins or polypeptides also include according to theinvention single or multiple substitutions, deletions and/or additionsof any molecules which are associated with the protein or polypeptide,such as carbohydrates, lipids and/or proteins or polypeptides.

In one embodiment, “derivatives” of proteins or polypeptides includethose modified analogs resulting from glycosylation, acetylation,phosphorylation, amidation, palmitoylation, myristoylation,isoprenylation, lipidation, alkylation, derivatization, introduction ofprotective/blocking groups, proteolytic cleavage or binding to anantibody or to another cellular ligand. Derivatives of proteins orpolypeptides may also be prepared by other methods such as, for example,by chemical cleavage with cyanogen bromide, trypsin, chymotrypsin,papain, V8 protease, NaBH₂, acetylation, formylation, oxidation,reduction or by metabolic synthesis in the presence of tunicamycin.

The term “derivative” also extends to all functional chemicalequivalents of proteins or polypeptides.

The derivatives, described above, of proteins and polypeptides areencompassed according to the invention by the term “fusion molecule”,even if no express reference is made thereto.

The pharmaceutical compositions described according to the invention canbe employed therapeutically for the treatment of a pre-existing diseaseor prophylactically as vaccines for immunization.

The term “vaccine” relates according to the invention to an antigenicpreparation which comprises for example a protein, a peptide, a nucleicacid or a polysaccharide, and which is administered to a recipient inorder to stimulate its humoral and/or cellular immune system against oneor more antigens which are present in the vaccine preparation. The terms“vaccination” or “immunization” relate to the process of administering avaccine and of stimulating an immune response against an antigen. Theterm “immune response” relates to the activities of the immune system,including activation and proliferation of specific cytotoxic T cellsafter contact with an antigen.

Animal models can be employed for testing an immunizing effect, e.g.against cancer on use of a tumor-associated antigen as antigen. It ismoreover possible for example, for human cancer cells to be introducedinto a mouse to create a tumor, and for a nucleic acid of the invention,which codes for a fusion molecule of the invention comprising thetumor-associated antigen, to be administered. The effect on the cancercells (for example reduction in tumor size) can be measured as criterionfor the efficacy of an immunization by the nucleic acid.

As part of the composition for immunization, one or more fusionmolecules are administered with one or more adjuvants to induce animmune response or increase an immune response. An adjuvant is asubstance which is incorporated into an antigen or is administeredtogether therewith and enhances the immune response. Adjuvants are ableto enhance the immune response by providing an antigen reservoir(extracellularly or in macrophages), activating macrophages andstimulating certain lymphocytes. Adjuvants are known and include in anonrestrictive manner monophosphoryl-lipid A (MPL, SmithKline Beecham),saponins such as QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham;WO 96/33739), QS7, QS17, QS18 and QS-L1 (So et al., Mol. Cells7:178-186, 1997), incomplete Freund's adjuvant, complete Freund'sadjuvant, vitamin E, Montanide, alum, CpG oligonucleotides (cf. Krieg etal., Nature 374:546-9, 1995) and various water-in-oil emulsions whichare prepared from biodegradable oils such as squalene and/or tocopherol.The fusion molecules are preferably administered in a mixture withDQS21/MPL. The ratio of DQS21 to MPL is typically about 1:10 to 10:1,preferably about 1:5 to 5:1 and in particular about 1:1. In a vaccineformulation for administration to humans, DQS21 and MPL are typicallypresent in a range from about 1 μg to about 100 μg.

Other substances which stimulate an immune response in the patient mayalso be administered. For example, cytokines can be used for avaccination because of their regulatory properties on lymphocytes. Suchcytokines include for example interleukin-12 (IL-12) which has beenshown to enhance the protective effects of vaccines (cf. Science268:1432-1434, 1995), GM-CSF and IL-18.

The method of the invention for inducing an immune response in a mammalgenerally comprises the administration of an effective amount of afusion molecule of the invention and/or of a nucleic acid codingtherefor, in particular in the form of a vector. DNA or RNA which codesfor a fusion molecule of the invention is preferably administered to amammal together with a DNA sequence which codes for a Tcell-costimulating factor, such as a gene coding for B7-1 or B7-2.

The expression “T cell-costimulating factor” relates herein to amolecule, in particular a peptide, which is able to provide acostimulating signal and thus enhances an immune response, in particularactivates the proliferation of T cells in the presence of one or morefusion molecules of the invention. Such an activation of T-cellproliferation can be determined by generally known assays.

These factors include costimulating molecules which are provided in theform of proteins or nucleic acids. Examples of such costimulatingmolecules are B7-1 and B7-2 (CD80 and CD86, respectively) which areexpressed on dendritic cells (DC) and interact with the CD28 moleculeexpressed on T cells. This interaction provides a costimulation (signal2) for an antigen/MHC/TCR-stimulated (signal 1) T cell, thus enhancingthe proliferation of the T cell and the effector function. B7 alsointeracts with CTLA4 (CD152) on T cells and investigations includingCTLA4 ligands and B7 ligands show that the B7-CTLA4 interaction canenhance an antitumor immunity and CTL proliferation (Zheng, P. et al.,Proc. Natl. Acad. Sci. USA 95(11):6284-6289 (1998)).

B7 is typically not expressed on tumor cells, so that they are noteffective antigen-presenting cells (APCs) for T cells. Induction of B7expression would make it possible for tumor cells more effectively tostimulate proliferation of cytotoxic T lymphocytes and an effectorfunction. Costimulation by a B7/IL-6/IL-12 combination showed aninduction of the IFN-gamma and Th1 cytokine profile in a T cellpopulation, leading to a further enhancement of T-cell activity(Gajewski et al., J. Immunol. 154:5637-5648 (1995)).

Complete activation of cytotoxic T lymphocytes and a complete effectorfunction requires cooperation of T-helper cells through the interactionbetween the CD40 ligand on the T-helper cells and the CD40 moleculewhich is expressed by dendritic cells (Ridge et al., Nature 393:474(1998), Bennett et al., Nature 393:478 (1998), Schonberger et al.,Nature 393:480 (1998)). The mechanism of this costimulating signalprobably relates to increasing the B7 and associated IL-6/IL-12production by the dendritic cells (antigen-presenting cells). TheCD40-CD40L interaction thus complements the interactions of signal 1(antigen/MHC-TCR) and signal 2 (B7-CD28).

The invention provides for administration of nucleic acids, polypeptidesor proteins and/or cells. Administration of DNA and RNA is preferred.

It was possible to show in the experiments that, compared with theunmodified antigen, according to the invention a 100-fold lower dose ofthe vaccine is sufficient to induce equivalent or stronger immuneresponses. One problem on direct injection of nucleic acid vaccines isthat the dose necessary to induce immune responses is very high. In thecase of DNA vaccines, the reason is presumably mainly based on the factthat only a fraction of the cells take up injected DNA into the nucleus.In the case of RNA vaccines, the problem is presumably that inparticular injected RNA is very rapidly degraded by RNAses.

It is to be expected on use of the vaccines modified according to theinvention that greatly increased immune responses will be obtained ondirect injection of nucleic acids, in particular RNA, compared withunmodified nucleic acids.

In a preferred embodiment, a viral vector for administering a nucleicacid which codes for a fusion molecule of the invention is selected fromthe group consisting of adenoviruses, adeno-associated viruses,poxviruses, including vacciniavirus and attenuated poxviruses, Semlikiforest virus, retroviruses, Sindbis virus and Ty virus-like particles.Adenoviruses and retroviruses are particularly preferred. Theretroviruses are normally replication-deficient (i.e. they are unable toproduce infectious particles).

Various methods can be employed according to the invention to introducenucleic acids into cells in vitro or in vivo. Such methods includetransfection of nucleic acid-calcium phosphate precipitates,transfection of nucleic acids associated with DEAE, transfection orinfection with the above viruses carrying the nucleic acids of interest,liposome-mediated transfection and the like. In particular embodiments,guiding of the nucleic acid to particular cells is preferred. In suchembodiments, a carrier employed for administering a nucleic acid to acell (e.g. a retrovirus or a liposome) may have a bound targetingmolecule. For example, a molecule such as an antibody which is specificfor a surface membrane protein on the target cell, or a ligand for areceptor on the target cell, can be incorporated into the nucleic acidcarrier or bound thereto. If administration of a nucleic acid byliposomes is desired, it is possible to incorporate proteins which bindto a surface membrane protein which is associated with endocytosis intothe liposome formulation in order to make targeting and/or uptakepossible. Such proteins include capsid proteins or fragments thereof,which are specific for a particular cell type, antibodies againstproteins which are internalized, proteins which target for anintracellular site, and the like.

The nucleic acids are preferably administered together with stabilizingsubstances such as RNA-stabilizing substances.

In one embodiment, the nucleic acids are administered by ex vivomethods, i.e. by removing cells from a patient, genetically modifyingthe cells, and reintroducing the modified cells into the patient. Thisgenerally includes the introduction of a functional copy of a gene intothe cells of a patient in vitro and returning the genetically modifiedcells to the patient. The functional copy of the gene is under thefunctional control of regulatory elements which permit expression of thegene in the genetically modified cells. Transfection and transductionmethods are known to the skilled worker. The invention also provides foradministration of nucleic acids in vivo through the use of vectors suchas viruses and targeted liposomes.

Administration of polypeptides and peptides can take place in a mannerknown per se.

The term “patient”, “individual” or “living creature” means according tothe invention a human, non-human primate or another animal, inparticular mammal such as cow, horse, pig, sheep, goat, dog, cat, birdssuch as chicken or rodent such as mouse and rat. In a particularlypreferred embodiment, the patient, the individual or the living creatureis a human.

The therapeutic compositions of the invention can be administered inpharmaceutically acceptable preparations. Such preparations can compriseusually pharmaceutically acceptable concentrations of salts, bufferingsubstances, preservatives, carriers, supplementary immunity-increasingsubstances such as adjuvants (e.g. CpG oligonucleotides) and cytokinesand, where appropriate, other therapeutic agents.

The therapeutic agents of the invention can be administered in anyconventional way, including by injection or by infusion. Theadministration can take place, for example, orally, intravenously,intraperitoneally, intramuscularly, subcutaneously, intracutaneously,transdermally, intralymphatically, preferably by injection into lymphnodes, especially inguinal lymph nodes, lymphatic vessels and/or intothe spleen.

The compositions of the invention are administered in effective amounts.An “effective amount” relates to the amount which, alone or togetherwith further doses, achieves a desired response or a desired effect. Inthe case of treatment of a particular disease or of a particularcondition, the desired response relates to inhibition of the progress ofthe disease. This includes slowing down the progression of the diseaseand in particular stopping the progression of the disease. The desiredresponse on treatment of a disease or of a condition may also bedelaying the onset or preventing the onset of the disease or of thecondition.

An effective amount of a composition of the invention depends on thecondition to be treated, the severity of the disease, the individualpatient's parameters, including age, physiological condition, height andweight, the duration of the treatment, the nature of a concomitanttherapy (if present), the specific administration route and similarfactors.

The pharmaceutical compositions of the invention are preferably sterileand comprise an effective amount of the therapeutically active substanceto generate the desired response or the desired effect.

The doses of the compositions of the invention which are administeredmay depend on various parameters such as the mode of administration, thepatient's condition, the desired administration period etc. In the casewhere a patient's response is inadequate with an initial dose, it ispossible to employ higher doses (or effectively higher doses which areachieved by a different, more localized administration route).

In general, doses of from 1 ng to 1 mg, preferably from 10 ng to 100 μg,of the tumor-associated antigen are formulated and administered for atreatment or for generating or enhancing an immune response. If it isdesired to administer nucleic acids (DNA and RNA), doses of from 1 ng to0.1 mg are formulated and administered.

The pharmaceutical compositions of the invention are generallyadministered in pharmaceutically acceptable amounts and inpharmaceutically acceptable compositions. The term “pharmaceuticallyacceptable” relates to a non-toxic material which does not interact withthe effect of the active ingredient of the pharmaceutical composition.Such preparations may usually comprise salts, buffering substances,preservatives, carriers and, where appropriate, other therapeuticagents. When used in medicine, the salts should be pharmaceuticallyacceptable. Non-pharmaceutically acceptable salts can, however, be usedto prepare pharmaceutically acceptable salts thereof and are encompassedby the invention. Such pharmacologically and pharmaceutically acceptablesalts include in a non-limiting manner those prepared from the followingacids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic,acetic, salicylic, citric, formic, malonic, succinic acids and the like.Pharmaceutically acceptable salts can also be prepared as alkali metalor alkaline earth metal salts such as sodium, potassium or calciumsalts.

A pharmaceutical composition of the invention may comprise apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” relates according to the invention to one or morecompatible solid or liquid fillers, diluents or capsule substances whichare suitable for administration to a human. The term “carrier” relatesto an organic or inorganic ingredient, natural or synthetic in nature,in which the active ingredient is combined in order to facilitate use.The ingredients of the pharmaceutical composition of the invention areusually such that no interaction which substantially impairs the desiredpharmaceutical activity occurs.

The carriers are preferably sterile liquids such as water or oils,including those derived from petroleum, animals or plants, or being ofsynthetic origin, such as, for example, peanut oil, soybean oil, mineraloil, sesame oil, sunflower oil and the like. Saline solutions andaqueous dextrose and glycerol solutions can also be used as aqueouscarriers.

Examples of excipients and carriers are acrylic and methacrylicderivatives, alginic acid, sorbic acid derivatives such asα-octadecyl-ω-hydroxypoly(oxyethylene)-5-sorbic acid, amino acids andtheir derivatives, especially amine compounds such as choline, lecithinand phosphatidylcholine, gum arabic, aromas, ascorbic acid, carbonatessuch as, for example, sodium, potassium, magnesium and calciumcarbonates and bicarbonates, hydrogen phosphates and phosphates ofsodium, potassium, calcium and magnesium, carmellose sodium,dimethicone, colors, flavorings, buffering substances, preservatives,thickeners, plasticizers, gelatin, glucose syrups, malt, colloidalsilicon dioxide, hydromellose, benzoates, especially sodium andpotassium benzoates, macrogol, skim milk powder, magnesium oxide, fattyacids and their derivatives and salts such as stearic acid andstearates, especially magnesium and calcium stearates, fatty acid estersand mono- and diglycerides of edible fatty acids, natural and syntheticwaxes such as beeswax, yellow wax and montan glycol wax, chlorides,especially sodium chloride, polyvidone, polyethylene glycols,polyvinylpyrrolidone, povidone, oils such as castor oil, soybean oil,coconut oil, palm kernel oil, sugars and sugar derivatives, especiallymono- and disaccharides such as glucose, fructose, mannose, galactose,lactose, maltose, xylose, sucrose, dextrose and cellulose and theirderivatives, shellac, starch and starch derivatives, especially cornstarch, tallow, talc, titanium dioxide, tartaric acid, sugar alcoholssuch as glycerol, mannitol, sorbitol and xylitol and their derivatives,glycol, ethanol and mixtures thereof.

The pharmaceutical compositions may preferably also comprise in additionwetting agents, emulsifiers and/or pH-buffering agents.

In a further embodiment, the pharmaceutical compositions may comprise anabsorption enhancer. These absorption enhancers may if desired replacean equimolar amount of the carrier in the composition. Examples of suchabsorption enhancers include in a non-limiting manner eucalyptol,N,N-diethyl-m-toluamide, polyoxyalkylene alcohols (such as propyleneglycol and polyethylene glycol), N-methyl-2-pyrrolidone, isopropylmyristate, dimethylformamide (DMF), dimethyl sulfoxide (DMSO),dimethylacetamide (DMA), urea, diethanolamine, triethanolamine and thelike (see, for example, Percutaneous Penetration Enhancers, edited bySmith et al. (CRC Press, 1995)). The amount of absorption enhancer inthe composition may depend on the desired effects to be achieved.

A protease inhibitor can be incorporated into the composition of theinvention in order to prevent degradation of a peptide or protein agentand thus to increase the bioavailability. Examples of proteaseinhibitors include in a non-limiting manner aprotinin, leupepsin,pepstatin, α2-macroglobulin and trypsin inhibitor. These inhibitors canbe used alone or in combination.

The pharmaceutical compositions of the invention can be provided withone or more coatings. The solid oral dosage forms are preferablyprovided with a coating resistant to gastric juice or are in the form ofa hardened soft gelatin capsule resistant to gastric juice.

The pharmaceutical compositions of the invention may comprise suitablebuffering substances such as acetic acid in a salt, citric acid in asalt, boric acid in a salt and phosphoric acid in a salt.

The pharmaceutical compositions may also comprise where appropriatesuitable preservatives such as benzalkonium chloride, chlorobutanol,parabens and thimerosal.

The pharmaceutical compositions are usually presented in a unit doseform and can be produced in a manner known per se. Pharmaceuticalcompositions of the invention may be for example in the form ofcapsules, tablets, lozenges, solutions, suspensions, syrups, elixirs oras emulsion.

Compositions suitable for parenteral administration usually comprise asterile aqueous or nonaqueous preparation of the active agent, which ispreferably isotonic with the recipient's blood. Examples of suitablecarriers and solvents are Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are usually employed asdissolving or suspending medium.

The present invention is described in detail by the following examplesand figures which serve exclusively for illustration and are not to beunderstood as limiting. Further embodiments which do not go beyond thebounds of the invention and the scope of the annexed claims areaccessible to the skilled worker on the basis of the description and theexamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Diagrammatic representation of a fusion protein of theinvention. The fusion protein consists of an N-terminally placedsecretion signal, of a C-terminally located transmembrane andcytoplasmic domain of a histocompatibility antigen, and of an integratedcomplete or partial sequence of an antigen.

FIG. 2: Diagrammatic representation of the cassettes for expression offusion proteins. SP: signal peptide; MCS: multiple cloning site; TM:transmembrane domain; MHC tail: cytoplasmic tail of an MHC molecule;antigen: sequence coding for an antigen against which immune responsesare to be induced.

FIG. 3: Testing of the effect of various RNA doses on the frequency ofantigen-specific CD4+ T lymphocytes.

1×10⁶ purified CD4+ lymphocytes were cocultivated for 1 week with 2×10⁵DC which had been transfected with RNA in the stated amounts (0.1-10 μgRNA) by electroporation. On day 7 after stimulation, an ELISPOT wascarried out under standard conditions to detect interferon-γ-secreting Tlymphocytes. The antigen-presenting cells used were DC from the samedonor which had been loaded with overlapping pp65 peptides (1.75 μg/ml)or an irrelevant control peptide. For the test, 3×10⁴ effectors werecoincubated with 2×10⁴ DC for 16 h. After standard development, thenumber of IFN-gamma-secreting T lymphocytes was determined by means of asoftware-based video analysis. Compared with the CMVpp65 standard RNA,there is seen to be a massive expansion of CD4+ lymphocytes both by theCMVpp65-TM1 construct and by the CMVpp65-TM2 construct.

FIG. 4: Testing of the effect of various RNA doses on the frequency ofinterferon-gamma-secreting CD8+ T lymphocytes. 1×10⁶ purified CD8+lymphocytes were cocultivated for 1 week with 2×10⁵ DC which had beentransfected with RNA in the stated amounts (0.1-10 μg RNA) byelectroporation. On day 7, a standard ELISPOT was carried out to detectIFN-gamma-secreting T lymphocytes against DC of the same donor which hadbeen loaded with overlapping pp65 peptides (1.75 μg/ml) or an irrelevantcontrol peptide. 3×10⁴ effectors were coincubated with 2×10⁴ DC for 16h. After standard development, the number of IFN-gamma-secreting Tlymphocytes was determined by means of a software-based video analysis.There was seen to be a massive expansion of CD8+ lymphocytes by theCMVpp65-TM1 construct and the CMVpp65-TM2 construct. Even on use of 100×lower doses (0.1 μg RNA), the frequency of the pp65-specific CD8+lymphocytes was still above the background after stimulation by DCtransfected with NYESO-RNA (data not shown). Stimulation by theCMVpp65standard construct showed an expansion of pp65-specificlymphocytes above the background level only with 2.5 μg and above.

FIG. 5: Dose/effect profile for the expansion capacity of variousimmunogens on antigen-specific lymphocytes. The immunogens modifiedaccording to the invention exhibit a distinctly increased potency(>100×) and a higher maximum effect.

FIG. 6: Comparative test of the effect of immunogens modified accordingto the invention and standard immunogens on the generation of cytotoxicimmune responses. 1×10⁶ purified CD8+ lymphocytes were cocultivated for1 week with 2×10⁵ DC which had been transfected with 10 μg of RNA byelectroporation. On day 7, a standard cytochrome cytotoxicity assayagainst DC of the same donor which had been loaded with variousconcentrations of overlapping pp65 peptides or an irrelevant controlpeptide was carried out. 15×10⁴ effectors were coincubated with 0.5×10⁴DC for 4 h. After measurement of the supernatant in a counter, thespecific lysis was calculated according to the formula: There was seento be extensive lysis by CD8+ lymphocytes which had been stimulated withCMVpp65-TM1 and CMVpp65-TM2 constructs, which was above the value forthe control peptide as far as a concentration of 10 nM of the pp65peptide mixture (data not shown). CD8+ lymphocytes were likewiseexpanded by the pp65 peptide mixture and showed a marked specific lysis,but did not reach the level of CMVpp65-TM1 and -TM2. Only a weakstimulation of pp65-specific cytotoxic T cells was achievable by theCMVpp65standard construct.

FIG. 7: Diagrammatic representation of the cassettes for expressingfusion proteins. CS: cloning site; TM: transmembrane domain; SNARE:SNARE protein or motif; antigen: sequence coding for an antigen againstwhich immune responses are to be induced.

FIGS. 8A-8E: Sequences used in the examples HLA class I TM-CM:transmembrane-cytoplasmic region of an HLA class I molecule; HLA classII TM-CM: transmembrane-cytoplasmic region of an HLA class II molecule.

FIG. 9: Sequences of transmembrane-cytoplasmic regions and cytoplasmicregions of MHC molecules. The sequences show thetransmembrane-cytoplasmic region or only the cytoplasmic region ofvarious HLA molecules. The transmembrane region is underlined and bold.

FIG. 10A-10C: Sequences of SNARE proteins. These sequences are suitablefor constructing the SNARE-antigen fusion molecules (N-SNARE-antigen) ofthe invention.

FIG. 11: Stimulation of naive CD8+ T lymphocytes by fusion constructs ofthe invention. In microtiter plates, 1×10⁵ CD8+ lymphocytes per wellwere stimulated against 2×10⁴ DC which were transfected with 20 μg ofCMVpp65-TM1 or control RNA. The medium was supplemented with IL-6 (1000U/ml) and IL-12 (10 ng/ml). On day +7 and +14, thawed transfected DC(2×10⁴/well) were used for restimulation, the medium containing IL-2 (10U/ml) and IL-7 (5 ng/ml). On day +21, all the populations were assayedin an ELISPOT against control peptides (1.75 μg/ml) and againstpp65-overlapping peptides (1.75 μg/ml). Two of the populationsstimulated against CMVpp65-TM1 (Pop.1, Pop.2) showed a marked pp65reactivity.

EXAMPLES Example 1 Preparation of the Modified Vaccines

To prepare the modified vaccines, firstly a cassette which permitsexpression of fusion genes was prepared in an expression vector whichpermits transcription of RNA. For this purpose, initially the nucleicacid which codes for a signal peptide of an HLA molecule was amplifiedfrom human lymphocytes, and the fragment was cloned as cDNA into avector (SEQ ID NO: 1 and 2). The cloning was carried out in such a waythat various restriction enzyme cleavage sites were located behind thecDNA of the signal peptide, and further fragments can be cloned in-framein the expression cassette. The selected vectors were plasmids whichpermit in vitro expression of RNA via a 5′-located RNA polymerasepromoter T3, T7 or SP6. The next fragment cloned into this vector was acDNA which encodes a transmembrane domain and the cytoplasmic domain ofan HLA class I (SEQ ID NO: 3 and 4) or class II (SEQ ID NO: 5 and 6)molecule, including stop codon. The cloning was carried out in such away that the resulting plasmid still has restriction enzyme cleavagesites for cloning antigens between the two fragments (SEQ ID NO: 7 and 8and FIG. 1). The sequence (SEQ ID NO: 9 and 10) coding for the humancytomegalovirus phosphoprotein 65 (pp65) was cloned into theseexpression cassettes as model antigen in such a way that a continuousORF composed of HLA signal sequence, pp65 and HLA transmembrane andcytoplasmic domain (SEQ ID NO: 11 and 12) resulted. A vector whichcomprised the pp65 sequence with a stop codon in the same initial vectorwithout said fragments was prepared for control experiments. Thefollowing nucleic acids were used for further experiments:

CMVpp65standard: unmodified CMVpp65 sequence, standard immunogenCMVpp65-TM1: fusion nucleic acid composed of the following fragments:HLA class I secretion signal, pp65 ORF and HLA class I transmembrane andcytoplasmic domain (modified immunogen).CMVpp65-TM2: fusion nucleic acid composed of the following fragments:HLA class I secretion signal, pp65 ORF and HLA class II transmembraneand cytoplasmic domain (modified immunogen).

Example 2 Testing of the Modified Vaccines

The three nucleic acids (CMVpp65standard, CMVpp65TM1, CMVpp65TM2) wereemployed as immunogen in stimulation tests with autologous DCs fromantigen-positive donors. In order to test CD4 and CD8 immune responsesseparately, purified CD4+ and CD8+ lymphocytes were used. The readoutemployed was the enzyme-linked immunospot assay (ELISPOT), which isacknowledged to be the standard assay for quantifying IFN-λ-secreting Tcells. A standard chromium release assay was used to assay the effectorfunction of CD8+T lymphocytes. Autologous monocytes or DCs weretransfected with pp65 RNA, CMVpp65-TM1 and CMVpp65-TM2 immunogens. DCswere loaded with overlapping peptides for pp65 and with control peptideas maximum stimulation control. The DCs treated in this way werecoincubated with CD4+ or CD8+ lymphocytes overnight or for 7 days. Thereadout took place against autologous monocytes or DCs which had beenpulsed with pp65 overlapping peptides or with a CMV fibroblast lysate.The investigation of CD4+ immune responses surprisingly revealed thatboth modified immunogens (CMVpp65-TM1 and CMVpp65-TM2) not only inducedan enhanced immune response to the CMVpp65standard immunogen, but alsoinduced a maximum level of antigen-specified IFN-gamma secretion in CD4+lymphocytes (FIG. 3). The percentage of antigen-specific CD4+ cellsafter stimulation by the modified pp65 constructs was moreover equal toor even higher than after stimulation with pp65 overlapping peptides. Asexpected, the CMVpp65standard immunogen showed no relevant stimulationof CD4+ lymphocytes.

An even more surprising result emerged on investigation of CD8 immuneresponses after stimulation with the immunogens. It was possible to showthat the use of the modified expression cassettes for stimulating CD8+lymphocytes likewise led to a proportion of specifically IFN-λ-secretingcells which is comparable to that after stimulation with pp65overlapping peptides. Surprisingly, the modified RNA constructs were farsuperior to the unmodified CMVpp65standard immunogens in this case too(FIGS. 4 and 5). The results in the cytotoxicity assay showed that bothmodifications led to a not previously described drastic increase incytotoxicity compared with CMVpp65standard RNA (FIG. 6). In this casetoo there was surprisingly seen to be a superiority of the modifiedimmunogens over the overlapping pp65 peptides.

Example 3 Stimulation of Naive CD8+ T Lymphocytes by HLA Fusion Antigens

In order to attest the possibility of priming and subsequent expansionof naive CD8+ lymphocytes by the fusion constructs of the invention,dendritic cells of a CMV-negative donor were transfected with RNA of theunmodified CMVpp65 or with CMVpp65-TM1 RNA or with a control RNA(NY-Eso-1). The transfected dendritic cells were employed to stimulateautologous CD8+ lymphocytes. 2 restimulations were carried out withfrozen transfected dendritic cells at weekly intervals. For the readout,on day +21 after the first stimulation, all cell populations wereassayed in an IFNγ ELISpot assay against autologous dendritic cellswhich were loaded either with pp65 overlapping peptides or, as control,with irrelevant overlapping peptides. It was found in this case thatpp65-reactive CD8+ T lymphocyte populations were generated bystimulation with CMVpp65-TM1 RNA in two cases (FIG. 11). Stimulationswith the dendritic cells transfected with the unmodified CMVpp65 RNA orwith control RNA by contrast showed no significant pp65 reactivity.

Example 4 Use of HLA Fusion Antigens for Stimulating Tumor Cell-ReactiveT Lymphocytes

In order to be able to expand CD8+ and CD4+ T lymphocytes againstdefined tumor antigens, the following antigen sequences were cloned asinserts into fusion constructs of the invention: the tumor antigen TPTE(Koslowski et al., 2004, PMID 15342378), the tumor antigen PRAME (Ikedaet al., 1997, PMID 9047241) in variant 1 (SEQ ID NO: 64), the tumorantigen WT1 as variant C (SEQ ID NO: 65) and the tumor antigen p53 (SEQID NO: 66). For the functional validation, human dendritic cells of anHLA* A 0201-positive donor were transfected either with WT1-HLA-TM1-RNA,with unmodified WT1-RNA or irrelevant control RNA and used as targetcells. After coincubation with WT1-reactive CD8+ T-cell clones for 8 or16 hours, IFNγ was quantified in the supernatant. It was seen thatsecretion was a factor of 6-9 higher after coincubation with WT1-HLA-TM1transfected dendritic cells by comparison with coincubation aftertransfection with unmodified WT1.

In a series of experiments, the following results were achieved insummary and confirmed several times:

-   -   The modified immunogens lead to a distinctly enhanced        stimulation and expansion of antigen-specific CD4+ lymphocytes        (increased proliferation of CD4+ lymphocytes)    -   The modified immunogens lead to a distinctly enhanced        stimulation and expansion of antigen-specific CD8+ lymphocytes        (increased proliferation of CD8+ lymphocytes)    -   The modified immunogens lead to a distinctly enhanced cytokine        release from antigen-specific CD4+ lymphocytes and CD8+        lymphocytes (increased cytokine release=increased activation)    -   The modified immunogens lead to a distinctly enhanced cytotoxic        reactivity of antigen-specific CD8+ lymphocytes (increased        cytotoxic effect)    -   The modified immunogens are 100× more potent in relation to the        expansion of antigen-specific CD8+ lymphocytes    -   The modified immunogens have, even at a 100× lower dose, a        stronger effect on the expansion of antigen-specific CD4+        lymphocytes than standard immunogens

In summary, therefore, it can be said that the modifications accordingto the invention of an antigen result in a more than 100-fold increasedpotency (leftward shift in the dose-effect curve) and a drasticallyincreased biological activity. Compared with the unmodified antigensequences customary to date, it is possible to generate an immunogenwhich has a quantitatively and qualitatively greater efficacy asvaccine.

An important result of the invention is that antigen-specific CD4+ andCD8+ lymphocytes are optimally stimulated and expanded simultaneously.Stimulation of CD8+ and CD4+ lymphocytes is crucially important for theefficacy in particular of therapeutic vaccines.

1. A fusion protein comprising an antigen, and a transmembrane regionand a cytoplasmic region of α chain of a MHC molecule or CD1a, CD1b orCD1c but free from (i) α1, α2 and α3 domains of the α chain of a MHCClass I molecule or CD1a, CD1b or CD1c, (ii) β2-microglobulin of a MHCClass I molecule or CD1a, CD1b or CD1c, (iii) α1 and α2 domains of the αchain of a MHC Class II molecule, and (iv) β1 and β2 domains of the βchain of a MHC Class II molecule.
 2. An isolated host cell whichcomprises the fusion protein as claimed in claim
 1. 3. A compositionwhich comprises the fusion protein of claim 1 in a pharmaceuticallyacceptable carrier.
 4. The fusion protein of claim 1, which includes anamino acid residue sequence selected from the group consisting of SEQ IDNO: 2, 4, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and
 42. 5. Thefusion protein of claim 1, which is encoded by a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO:
 13. 6.The fusion protein of claim 1 which includes the amino acid residuesequence of SEQ ID NO:
 12. 7. The fusion protein of claim 1 which isencoded by a nucleic acid molecule comprising the nucleotide sequence ofSEQ ID NO:
 11. 8. The fusion protein of claim 1 which is encoded by anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:13.
 9. The fusion protein of claim 1 comprising the amino acid residuesequence of SEQ ID NO:
 14. 10. A composition which comprises theisolated host cell of claim
 2. 11. The fusion protein of claim 1 whereinthe antigen is a tumor antigen or a viral antigen.
 12. The fusionprotein of claim 11 wherein the tumor antigen is selected from the groupconsisting of carcinoembryonic antigen, α1-fetoprotein, isoferritin,fetal sulfoglycoprotein, α2-H-ferroprotein, and γ-fetoprotein and theviral antigen is selected from the group consisting of a viralribonucleoprotein and a viral envelope protein.
 13. The fusion proteinof claim 1 wherein the transmembrane region and the cytoplasmic regiontogether comprise an amino acid residue sequence selected from the groupconsisting of SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, and
 41. 14. The fusion protein of claim 1 wherein the cytoplasmicregion comprises an amino acid residue sequence selected from the groupconsisting of SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, and
 42. 15. The fusion protein of claim 1 wherein the fusion proteinhas the amino acid residue sequence of SEQ ID NO:
 14. 16. The fusionprotein of claim 1 wherein the antigen is selected from the groupconsisting of ART-4, BAGE, ss catenin/m, Bcr-abL CAMEL, CAP-1, CASP-8,CDC27/m, CDK4/m, CEA, claudin-12, c-MYC, CT, Cyp-B, DAM, ELF2M,ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2/neu, HPV-E7, HPV-E6,HAST-2, hTERT, hTRT, LAGE, LDLR/FUT, MAGE-A, MAGE-A1, MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, MAGE-B, MAGE-C, MART-1/melan-A, MC1R, myosin/m,MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minorbcr-abL Pml/RARa, PRAME, proteinase-3, PSA, PSM, RAGE, RU1 or RU2, SAGE,SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, survivin, TEL/AML1,TPI/m, TRP-1, TRP-2, TRP-2/INT2, TPTE, and WT.
 17. The fusion protein ofclaim 1 comprising an amino acid residue sequence selected from thegroup consisting of SEQ ID NO: 2, 4, 12, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, and
 42. 18. The fusion protein of claim 1, wherein the fusionprotein additionally comprises a leader sequence.
 19. The fusion proteinof claim 18, wherein the leader sequence is derived from an MHCmolecule.
 20. The fusion protein of claim 18, wherein the fusion proteinhas the following arrangement of segments: N terminus—leadersequence/antigen/transmembrane region/cytoplasmic region—C terminus, andthe individual segments optionally are separated from one another bylinker sequences.
 21. The fusion protein of claim 18, wherein the leadersequence comprises the amino acid sequence of SEQ ID NO: 2.